Process for the Production of Ambrafuran

ABSTRACT

A method for the cyclodehydration of a 1,4- or 1,5-diol includes the step of exposing a 1,4- or a 1,5-diol to an activated zeolite at a temperature of between about 0° C. and about 110° C. for a period of between about 1 and 24 hours. The activated zeolite is prepared from an inactive NaY or CaY type zeolite by ion exchange with an ammonium salt, to produce an ammonium zeolite and exchange of at least part of the ammonia of the ammonium zeolate with a group II A metal.

This application claims the benefit of priority of South AfricanProvisional Patent Application No. 2009/02099, filed on Mar. 25, 2009,the disclosure of which is incorporated herein by reference in itsentirety.

THIS INVENTION relates to the dehydration of alcohols. It relates inparticular to the cyclodehydration of dials and to a process for theproduction of ambrafuran.

The food, feed, cosmetic, chemical and pharmaceutical sectors makeextensive use of flavours and fragrances. Although many commerciallyavailable flavour compounds are produced via chemical synthesis orthrough extraction from plant and animal sources, there is a movement toproduce these active compounds via bio-production which includesfermentation or bio-conversions using biocatalysts. The reason for thisis partly because of consumer demand for “green products” which aremanufactured by environmentally friendly chemical processes and partlybecause normal synthetic processes generally produce racemic mixturesinstead of single enantiomers. The isolation of active compounds fromplant and animal sources also usually has the drawback that thesecompounds are present in small quantities and this results in expensiveprocesses.

For centuries, ambergris has been a very valuable perfumery material andhas been used as a fixative agent in perfumes. A fixative agent, whichcan be a natural or a synthetic compound, reduces the rate ofevaporation of volatile substances in perfumes and stabilises perfumes.Ambergris is a metabolic product produced by the sperm whale (Physetermacrocephatus L.). Ambergris is formed in the rectum of the whale fromindigestible objects from the animals on which it feeds. These usuallyinclude the beaks of squid and cuttlefish, and the ambergris is normallyreleased when the whale dies. Ambergris contains a large amount ofsteroid lipids and has a lower density than water. Following initialrelease, the ambra which is a pathological metabolite of the sperm whaleis soft and pale white and has a strong manure smell. During exposure tothe elements at sea, the ambra is oxidised and it loses the strongoffensive smell and the characteristic ambergris odour develops. Thematerial (−)-ambrafuran is the most important and sought-after of thecompounds of the ambergris type and is marketed by Firmenich S. A underthe trade mark Ambrox®. The literature names for (−)-ambrafuran aredodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan or(−)-8,12-epoxy-13,14,15,16-tetranorlabdane.

Several routes have been developed to produce (−)-ambrafuransynthetically and many are based on naturally occurring sesqui- orditerpenes. Sclareol has been used in industrial processes for thesynthesis of (−)-ambrafuran as it has the same stereochemical featuresas (−)-ambrafuran. It has been reported that any changes in theconfiguration of the four chiral carbon atoms of ambrafuran have majoreffects on the odour, the profile and the strength of the compound.

The synthesis of (−)-ambrafuran from sclareol in an overall yield ofapproximately 76% has been reported and describes some of the problemsencountered with the synthesis of ambrafuran from sclareol including thehemisynthesis of ambrafuran from sclareol which was carried out in threestages and in eight steps. These involved the oxidative degradation ofthe sclareol side chain to yield sclareolide and its acetoxy-acidprecursor, followed by the reduction of the intermediate compounds toambradiol and a final cyclodehydration to yield (−)-ambrafuran. Thefirst stages of the synthesis appear to be problematic as threeintermediates have to be formed successively. Potassium permanganate (7moles per mole sclareol) was normally used for the oxidation of sclareoland only gave a yield of 65% sclareolide following saponification andlactonisation of the intermediate acetoxy-acid. A by-product of thereaction, was the production of manganese dioxide in quantities almostdouble the weight of the sclareol. This was a sticky solid and which wasvery difficult to remove. To overcome this problem, the manganesedioxide was converted to water-soluble manganese salts by reductionunder acidic conditions using sulphur dioxide, sodium hydrogen sulfiteand oxalic acid. This however resulted in aqueous waste disposalproblems. Ruthenium tetraoxide (normally prepared in situ from rutheniumchloride), used in combination with common oxygen donors, is also usedfor catalytic oxidation. Prior art methods include the reaction ofsclareol with ruthenium chloride hydrate (0.023 mol/mol) and sodium (˜8periodate mol/mol) under typical Sharpless conditions to give a mixtureof the acetoxy-acid and sclareolide in an 88.5% overall yield. Inanother prior art method the sodium periodate and organic solvent werereplaced with sodium hypochlorite and water respectively and the yieldof the sclareolide after saponification and lactonisation of theacetoxy-acid was 75-78%.

Other researchers have described the conversion of ambradiol to(−)-ambrafuran by ring closure through cyclodehydration. It has beenshown that if the ring closure involves the attack on the 12-carbon bythe 8-oxygen, the configuration of the 8-chiral carbon should beretained. This normally happens when the common method for preparationof cyclic ethers from 1,4-diols using tosyl chloride in pyridine isused. The reaction involves displacement of the tosyloxy group, which isselectively formed from the primary 12-hydroxyl, by the 8-oxygen in anucleophilic substitution process. It may however be advantageous toavoid the use of pyridine entirely since, even at low levels, pyridinemay interfere with the fragrance of (−)-ambrafuran. It has been reportedthat pyridine can be successfully replaced by other basic compounds,such as sodium hydride and sodium tert-amyl alcoholate. It has also beenreported that (−)-ambrafuran can be obtained in 96% yield (75% overallfrom sclareol) by reacting the diol with butyllithium and tosylchloride.

Another prior art method describes the carbonylation of allylic alcoholsin the synthesis of an ambergris fragrance compound. Relevant in thisprocess is the final step of the synthesis which involves thecyclisation to (−)-ambrafuran. The appropriate alcohol can be treatedwith a Lewis or Brönsted acid to achieve the cyclisation. A wide varietyof acidic reagents were found to be able to result in thetransformation. These reagents include boron trihalide and complexesthereof and several sulfonic acids. Trifluoromethanesulfonic acid, borontrifluoride and its complexes as well as alkyl- or arylsulfonic acidsseem to be the preferred catalysts. The preferred solvents in which tocarry out the cyclisation reaction seem to be halocarbon solvents attemperatures which vary from −110° C. to 150° C. At least 1, but up to 5molar equivalents of the acidic cyclisation reagent need to be used.

U.S. Pat. No. 3,029,255 describes a method for achieving the cyclisationto (−)-ambrafuran by treatment ofdecahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol with Al₂O₃ at200-225° C., followed by heating in vacuo in the presence ofβ-naphthalene sulfonic acid (130-160° C.).

The synthesis of (−)-ambrafuran therefore involves approximately eightsteps, some of which require the use of very harsh reagents which haveto be disposed of in a very careful manner. FIG. 1 shows the conversionof sclareol to ambrafuran according to the prior art.

Zeolites are aluminosilicates which are generally used as solid acidcatalysts. Because of their channel dimensions and stable structuresthese materials show unique selectivity and reactivity. Zeolites areenvironmentally benign and their use generally results in a reduction inwaste and pollution. Zeolites, alumina and montmorillonite clay havebeen used for the catalytic dehydration of monoalcohols to ethers andolefins. The cyclodehydration of diols has also been used for thesynthesis of heterocyclic compounds. Most of the cyclodehydrationreactions using zeolites reported in the literature use very hightemperatures (of the order of 175-225° C.). The present invention on theother hand provides a novel method for the cyclodehydration of a diolusing a zeolite at room temperature with a total conversion of the diolto the cyclodehydrated product generally in less than two hours.

According to a first aspect of the invention there is provided a methodfor the cyclodehydration of a 1,4- or 1,5-diol, the method including thestep of exposing a 1,4- or a 1,5-diol to an activated zeolite at atemperature of between about 0° C. and about 110° C. for a period ofbetween about 1 and 24 hours, the activated zeolite being prepared froman inactive NaY type zeolite by ion exchange with an ammonium salt, toproduce an ammonium zeolite and exchange of at least part of the ammoniaof the ammonium zeolate with a group II A metal.

The NaY type zeolite may be that obtained from Zeolyst International(CBV100). Alternatively the calcium type zeolite (CBV320A) also obtainedfrom Zeolyst International may be used.

In the case of the NaY-type zeolite, ion exchange with the ammonium saltis preferably carried out until the sodium level has been reduced¹. TheGroup II A metal is preferably calcium. The ion exchange of the ammoniumcations with calcium is preferably carried out using calcium nitrate,although any suitable calcium salt may be used.

The cyclodehydration reaction may be carried out in a solvent such astoluene, ethyl acetate, diethyl ether, tetrahydrofuran or hexane.

In a preferred embodiment of the invention, the reaction may be carriedout in a hydrocarbon or aromatic hydrocarbon solvent such as hexane ortoluene at room temperature over a period of about 1 to 24 hours. OtherC₅-C₉ hydrocarbon or aromatic hydrocarbon solvents may also be used.

The diol may be tetranorlabdane diol (or amdradiol). The product of thecyclodehydration may then be (−)-ambrafuran or Ambrox®.

According to a second aspect of the invention, there is provided amethod of synthesing (−)-ambrafuran, the method including themicrobiological conversion of sclareol to ambradiol followed bycyclodehydration to produce ambrafuran.

The microbiological conversion of the sclareol to ambradiol may beconducted with the micro organism Hyphozyma roseoniger.

The conversion of sclareol to a diol intermediate using themicroorganism Hyphozyma roseoniger was described in 1989². Themicroorganism has the identifying characteristics of CBS214.83 and ATCC20624. The organism was cultivated under aerobic conditions in anaqueous nutrient medium. Different forms of the organism could be usedto achieve the conversion. These ranged from using the culturesuspension, i.e. including the cells and the corresponding nutrientsolution, or as suspended cells in a buffer solution, or even byimmobilising the cells or an enzyme extract thereof on a solid support.

The aqueous medium for growing the organism may contain nitrogensources, inorganic salts, growth factors, the desired substrate andadditional carbon sources. When small amounts of yeast extract wereadded, supplementation with vitamins and trace minerals was notnecessary. One or more trace minerals such as Fe, Mo, Cu, Mn and B couldbe added as well as vitamin B complex. The preferred temperature rangefor the cultivation of the microorganism was between about 18 and 28° C.with a pH between 3 and 6.5. The substrate range could vary between 1.5and 30 g/l for optimum transformation. The substrate could be added tothe medium as a powder or in the presence of an emulsifier such as Tween80, as a slurry, or as a solution in an organic solvent such as acetone,ethanol or methanol. The organism was isolated from a soil sample fromcentral New Jersey in the USA and deposited with CBS (Centraalbureauvoor Schimmel Cultures) as well as with the ATCC (American Type CultureCollection).

The cyclodehydration step may be carried out using a Group II A metalzeolite. The Group II A metal may be calcium. The calcium zeolite may bea zeolite as described above. Instead the cyclodehydration step may becarried out in a hydrocarbon or aromatic hydrocarbon solvent such ashexane or toluene at room temperature or by dissolving ambradiol in asolvent such as dimethylsulphoxide (DMSO) or ethylacetate and optionallyheating the solution.

For example the cyclodehydration may be conducted in DMSO at atemperature of between about room temperature and 180° C. Alternativelythe cyclodehydration may be conducted in ethyl acetate at temperature ofbetween about −20° C. to about 37° C.

The cyclodehydration step produces the (−)-isomer of ambrafuran i.e.Ambrox®. The starting material (sclareol) is a racemic mixture and theapplicant believes that the microbiological oxidation of the racemicsclareol may be enantiomerically selective and produces a singleenantiomer of ambradiol. However, the applicant has not ruled out thepossibility that the desired enantiomer may be produced during thecyclodehydration step.

The invention is now described, by way of example, with reference to thefollowing examples and the Figures, in which

FIG. 1 shows a reaction scheme for the synthesis of (−)-ambrafuran fromsclareol;

FIG. 2 shows a reaction scheme for the synthesis of (−)-ambrafuran fromsclareol using the microorganism Hyphozyma roseoniger.

EXAMPLE 1 Analytical Methods (1) Quantitative Analysis of theIntermediate Diol and Ambrafuran

A Restek Rtx-5 sil w/intergra Guard, 0.25 mm ID. 0.25 μm film thickness(df) 30 meter GC column was used to analyse the conversion of thesclareol to ambradiol and the diol to ambrafuran. The GC program startedat 180° C. and was increased to 270° C. at a rate of 15° C. per minutewith a final run time of 6 minutes. The ambrafuran had a peak at 2.3minutes, the diol at 3.6 minutes and the sclareol at 4.5 minutes.Calibration curves for the diol and (−) ambrafuran were alsoconstructed.

(2) Chiral separation of (+) and (−)-ambrafuran

A Restek Rt-βDexsm 0.32 mmID. 0.25 μm df, 30 meter length was used toseparate (+) and (−) ambrafuran. The temperature was held constant at145° C. for 20 minutes. The (+) ambrafuran peak was at 17.3 minutes andthe (−) ambrafuran at 16.42 minutes.

The structures of the diol and ambrafuran were confirmed by LC-MS

EXAMPLE 2 Production of the Intermediate Diol from Sclareol UsingHyphozyma roseoniger Reconstituting the Microorganism

The Hyphozyma roseoniger was purchased from ATCC in a freeze-driedpowder form. It was reconstituted in sterile water and inoculated ontoagar plates. The agar plates consisted of potato dextrose agar and theyeast cultivation medium. The plates were grown for 4 days at roomtemperature. The microorganism was streaked onto another set of platesto ascertain purity of the culture. It was then inoculated into brothconsisting of the yeast cultivation medium. It was grown for 3 days andthe cells were spun down and re-suspended in a minimal volume of 100 mMpotassium phosphate buffer, pH6.5. The cell suspension was mixed with anequal volume of 50% glycerol and then placed into cryovials as themaster cell bank.

All experiments were carried out using a vial from the master cell bank.Microorganism (500 μl) was inoculated into 10 ml of either potatodextrose broth (PDB), the PDB plus substrate (sclareol), malt extract ormalt extract plus substrate, nitrogen base or nitrogen base plussubstrate. The cultures were grown for 3 days at room temperature andagitation at 180 rpm.

The microorganism (5 ml into 100 ml) was then inoculated into differentmedia containing substrate (0.02%). The different media could beselected from nitrogen base, potato dextrose broth plus nitrogen baseand nitrogen base plus malt extract.

In one set of experiments, the microorganism was first grown in potatodextrose broth plus nitrogen base or malt extract and nitrogen base for3 days without substrate. The cells were harvested and then re-suspendedin 100 mM potassium phosphate buffer pH 6.5 and the substrate was added.

Samples were taken every 24 hours for 5 days and analysed for theformation of the intermediate diol and any unwanted peaks.

Different substrate concentrations were also tested ranging from 10mg/100 ml to 20 g/100 ml.

To improve the substrate concentration for maximum productivity, a setof experiments was done in which the microorganism was grown in yeastnitrogen base without amino acids containing the substrate (0.02%) andTween 80 (500 μl/100 ml). Substrate (0.5 g) mixed with 0.5 g of Tween 80was then added every 24 hours for 5 days and the conversion to diol wasmonitored.

The preferred conditions were to grow the microorganism as normal for 3days with 0.02% substrate and then 1 g of substrate mixed with 1 g ofTween 80/100 ml was added and monitored for 8 days for conversion.Scaled-up reactions were carried out in which 10 g to 15 g of sclareoland 10 ml Tween 80 were added to a 1 L reaction mixture. The preferredtemperature for the conversion of sclareol to intermediate diol was 20°C. The temperature range was between 18° C. and 32° C.

Following full conversion of the sclareol to the diol, the diol wasextracted from the mixture by addition of ethyl acetate, separated fromthe aqueous phase and dried over anhydrous magnesium sulphate and thesolvent removed under reduced pressure.

The media described above for cultivating the microorganism as well asconverting the substrate all gave good conversion, but the yeastnitrogen base without amino acids with substrate gave the intermediatediol (>98% yield) without any by-products.

EXAMPLE 3 Preparation of the Zeolites

Inactive zeolites NaY type from Zeolyst (25 g) was mixed with 250 ml 10%ammonium nitrate and ion exchanged by refluxing at 90° C. for 24 hours.The mixture was filtered, washed with distilled water and driedovernight at 105° C. The procedure was repeated with 10% calcium nitrateand the zeolite was then activated at approximately 500° C. undervacuum.

Alternatively the Zeolite CBV320 (CaY type) can be purchased fromZeolyst International in the inactivate form and can be activated undervacuum at 500° C.

Yet another alternative was to activate the Zeolite CBV320 in aconventional microwave oven. The preferred method was to activate 50 gof Zeolite CBV320 by heating at 500 W for 15 minutes in an opencontainer in the microwave. The Zeolites were allowed to cool and thenkept in a closed container

EXAMPLE 4 Conversion of the Diol to (−)Ambrafuran

The conversion of the diol to the (−) ambrafuran was accomplished bycyclodehydration. Approximately 10 to 50 mg of the intermediate diol,prepared from sclareol with the Hyphozyma roseoniger microorganism asdescribed above was, in different embodiments of the invention,dissolved in 10 ml of toluene, ethyl acetate, diethyl ether, ethanol orhexane and placed in a round bottom flask. Activated zeolite (10 mg to500 mg), prepared as described above, was added and the mixture wasallowed to react at temperatures ranging from room temperature to 110°C. for 1 to 24 hours. The results are set out in Table 1 below.

TABLE 1 Conversion of the diol to (−) ambrafuran with different solventsin 4 hours at room temperature Solvent Conversion (4 hours) Diethylether 25 Ethanol 5.4 Ethyl acetate 3.7 Toluene 100 Hexane 100

In another embodiment of the invention, the reaction was carried out atroom temperature for 1 to 4 hours in toluene with a ratio of 400 mg diolto 20 ml toluene and 1:4 to 1:9 diol to activated zeolite. With thetoluene and activated zeolite, full conversion was achieved in 1 to 24hours at room temperature without the formation of any by-products. Theproduct in each case was the (−)-enantiomer, (−)-ambrafuran, as shown byGC. The applicant believes that the (−)-enantiomer is produced in themicrobiological conversion of racemic sclareol by the Hyphozymaroseoniger to produce an optically active diol².

The preferred method was to dissolve 400 mg of the intermediate diol in20 ml hexane with 1.6 to 3.6 g (1:4 to 1:9 ratio) of activated CBV320zeolites. The mixture was allowed to react at room temperature for 2 to24 hours. The zeolites were removed with centrifugation at 3000 rpm for5 minutes. The zeolites were washed with warm hexane or warm ethanol toremove any product associated with the zeolites. The hexane was removedunder reduced pressure to yield a final product (−) Ambrafuran with apurity of at least 96% and yield of 98%.

The reaction could be conducted with or without a nitrogen blanket. Thezeolite was filtered off or the mixture centrifuged to remove thezeolite and the solvent was removed under reduced pressure.

In other embodiments, the cyclodehydration was carried out in DMSO.Approximately 10 mg of the diol was dissolved in 10 ml DMSO dried onmolecular sieves (4 Å). In different embodiments, the reaction was runat temperatures ranging from room temperature to 180° C. under anitrogen blanket. In a preferred embodiment, the temperature was about180° C.

In other embodiments the diol was dissolved in ethyl acetate attemperatures from between −20° C. and 37° C. for approximately 2 weeks.This also resulted in conversion of the diol to the (−)-ambrafuran.

DISCUSSION

The reaction accordingly provides a novel process for thecyclodehydration of ambradiol to (−)-ambrafuran using activated zeolites(activated at 500° C. under vacuum or in a conventional microwave) atroom temperature using a hydrocarbon solvent such as hexane or toluene.It also provides a novel process for producing (−)-ambrafuran fromambradiol in a cyclodehydration using DMSO at elevated temperatures. Italso provides a method of producing (−)-ambrafuran from racemic sclareolby microbiological conversion of sclareol to ambradiol using Hyphozymaroseoniger followed by cyclodehydration to (−)-amberfuran. The inventionfurther provides an activated zeolite by activation of an inactivezeolite (NaY type) by ion exchange with ammonium nitrate followed by ionexchange with calcium nitrate followed by high temperature drying or useof the calcium zeolite CBV320. The zeolites used in the method of theinvention can be re-activated merely by heating at 500° C. under vacuumor at 500 W in a conventional microwave oven.

The invention thus provides a new process for the cyclodehydration of adiol precursor for the synthesis of the ambergris compound(−)-ambrafuran as well as an efficient two step process for the completesynthesis of (−)-ambrafuran from sclareol by the conversion of sclareolto an intermediate diol using a microorganism and cyclodehydration ofthe intermediate diol to (−)-ambrafuran.

REFERENCES

-   (1) Y Kanno, Y Matsui, H Imai (1985). Mechanistic model of    disproportionation of nitrogen monoxide on CaHY-type sodium. journal    of Inclusion Phenomena 3, 461-469.-   (2) M I Farbood, B J Willis (1989). Process for producing diol and    furan and microorganism capable of same. U.S. Pat. No. 4,798,799.

1. A method for the cyclodehydration of a 1,4- or 1,5-diol, the methodincluding the step of exposing a 1,4- or a 1,5-diol to an activatedzeolite at a temperature of between about 0° C. and about 110° C. for aperiod of between about 1 and 24 hours, the activated zeolite beingprepared from an inactive NaY or CaY type zeolite by ion exchange withan ammonium salt, to produce an ammonium zeolite and exchange of atleast part of the ammonia of the ammonium zeolate with a group II Ametal.
 2. A method as claimed in claim 1, in which the Group II A metalis calcium.
 3. A method as claimed in claim 1, in which the ion exchangeof the ammonium cations with calcium is carried out using calciumnitrate.
 4. A method as claimed in claim 1, in which thecyclodehydration reaction is carried out in a solvent selected fromtoluene, ethyl acetate, diethyl ether, tetrahydrofuran, hexane andmixtures thereof.
 5. A method as claimed in claim 4, in which thereaction is carried out in toluene at room temperature over a period ofbetween about 1 and 24 hours.
 6. A method as claimed in claim 4 in whichthe reaction is carried out in hexane at room temperature over a periodof between 1 to 24 hours.
 7. A method as claimed in claim 1, in whichthe diol is tetranorlabdane diol (or amdradiol).
 8. A method ofsynthesing (−)-ambrafuran, the method including the microbiologicalconversion of sclareol to ambradiol followed by cyclodehydration toproduce ambrafuran.
 9. A method as claimed in claim 8, in which themicrobiological conversion of the sclareol to ambradiol is carried outusing the micro organism Hyphozyma roseoniger.
 10. A method as claimedin claim 8, in which the cyclodehydration step is carried out using aGroup II A metal zeolite.
 11. A method as claimed in claim 10, in whichthe Group II A metal is calcium.
 12. A method as claimed in claim 8, inwhich the cyclodehydration step is carried out by dissolving ambradiolin a solvent and optionally heating the solution.
 13. A method asclaimed in claim 12, in which the solvent is selected from hydrocarbonand aromatic hydrocarbon solvents and the reaction is carried out atroom temperature.
 14. A method as claimed in claim 13, in which thehydrocarbon and aromatic hydrocarbon solvents are selected from hexaneand toluene.
 15. A method as claimed in claim 12, in which the solventis selected from dimethylsulphoxide (DMSO) and ethylacetate.
 16. Amethod as claimed in claim 15, in which the cyclodehydration isconducted in DMSO at a temperature of between about room temperature and180° C.
 17. A method as claimed in claim 15, in which thecyclodehydration is conducted in ethyl acetate at a temperature ofbetween about −20° C. and 37° C.